Opioid drugs and peptides act through multiple receptors and second messenger systems, but the classical narcotic analgesics, such as morphine, interact preferentially with the mu opioid receptor. We have identified a human neuroblastoma line (SK-N-SH) that expresses abundant mu opioid receptors (50,000 sites per cell) and delta receptors (10,000 sites per cell). It represents the only mu receptor cell line currently available that can be grown continuously in tissue culture. Differentiation with retinoic acid (RA) of the neuroblastic subclone. SH-SY5Y, causes neuronal differentiation and greatly enhances opioid sensitivity, while treatment with a combination of RA and herbimycin-A yields a homogeneous neuronal culture and increases mu opioid receptor content. Optimal cell culture conditions will be defined to provide a unique model for studying the opioid receptor and its interaction with narcotic analgesics. The fully developed in vitro model will serve to determine the mechanism of opioid receptor interaction with GTP binding proteins (Gi and Go) and two second messenger systems that are opioid responsive in this line (adenylate cyclase and Ca++) important questions include the relative contributions of mu and delta receptors in regulating these processes, possible interactions between mu and delta opioid receptors, mechanisms of tolerance, such as receptor down-regulation, receptor desensitization and phosphorylation, loss of G proteins, and biochemical correlates of dependence, i.e., the cAMP overshoot. Opioid receptors will be purified from high yield cultures to obtain partial sequences from peptide fragments and to determine their post-translational modification, e.g., by phosphorylation as a potential mechanism of desensitization. Novel techniques of high molecular weight tandem mass spectrometry will be employed for peptide analysis, which also permit detailed analysis of phosphorylation, glycocylation, and acylation of the receptor protein. Further, tolerance and cross-tolerance will be studied between the opioid receptors and the alpha-2 adrenergic receptor after transfection of the alpha-2 R gene into SH-SY5Y cells. The results will address the question of homologous and heterologous desensitization of adenylate cyclase inhibition by morphine and clonidine which suppresses opioid withdrawal symptoms. In order to gain further insights into the molecular nature of the opioid receptor, we will also attempt to identify the genes encoding the opioid receptors, either from partial peptide sequences or with the use of a mammalian expression vector cloning system. Upon availability of opioid receptor genes in suitable vectors, stably transfected cell lines will be established to study receptor mechanisms under selected conditions with specified opioid receptor content, and mutations will be introduced to determine the functional domains of the opioid receptor.